PROJECT SUMMARY Cytochrome P450s (P450s) are oxidases involved in a wide variety of synthetic and metabolic biochemical reactions in organisms throughout the kingdoms of life. Importantly, P450s are the central enzymes of drug metabolism. Despite similar structures and catalytic mechanisms, P450 homologs vary greatly in their specificity for and catalytic selectivity on different substrates, and these differences can have critical therapeutic consequences. A major obstacle in predicting P450 reactivity on any given substrate is our incomplete understanding of the involvement of protein dynamics - the population of multiple states and their interconversion - in the mechanisms of regioselectivity. Specifically, the population of multiple bound states could orient multiple parts of the substrate with respect to the reactive intermediate compound I and lead to multiple products. In addition, flexibility of the active site within one bound state could permit multiple positions of the substrate to approach the reactive compound I intermediate during its lifetime. Experimentally testing these possibilities is however challenging due to the complex, heterogeneous nature of the proteins and the contribution of motion on very fast timescales to protein flexibility. Infrared (IR) spectroscopy can resolve protein conformations and dynamics that interconvert on even the fastest timescales and, furthermore, 2D techniques can quantify conformational heterogeneity, as well as the frequency fluctuation amplitudes and timescales with which they are sampled. When combined with the spatial precision provided by site-selective labeling with IR probes, the approach should enable unprecedented description of the energy landscapes of P450s. This application is directed at three P450s which vary in flexibility: P450cam, 3A4, and 2C9 and their complexes with substrates that are hydroxylated with differing levels of regioselectivity. Measurement and comparison of the dynamics of the substrate complexes will provide information for evaluating how dynamics are involved in their different activity. The new information about P450s will advance our understanding of the biophysical mechanisms that underlie enzyme function, as well as improve our ability to predict P450 activity on a given molecule, and thus develop better drugs with improved pharmacokinetics.